Composition and method for dermal and transdermal administration of a cytokine

ABSTRACT

A composition for transdermal administration of a cytokine is described. The composition includes a conjugate composed of a cytokine, such as an interferon, and at least one fatty acid moiety covalently attached to the cytokine. The conjugate has enhanced cutaneous delivery relative to the cytokine alone.

[0001] This application claims the priority of U.S. ProvisionalApplication Ser. No. 60/068,873, filed Dec. 26, 1997, and which isincorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a composition for transdermaladministration of a cytokine. The composition includes a conjugatecomposed of a cytokine and at least one fatty acid moiety covalentlyattached to the cytokine.

BACKGROUND OF THE INVENTION

[0003] The routine administration of therapeutic proteins and peptidesis hindered by the lack of a reliable and convenient mode of delivery.The oral route is often impractical due to the digestion of proteins inthe gastrointestinal tract. Parenteral administration is an alternative,although frequent injections are required due to the short half-life ofpeptides and this can decrease patient compliance.

[0004] Other potential routes of administration for proteins includenasal, pulmonary, rectal, vaginal, ocular and transdermal. Thetransdermal route offers some advantages in that the skin has lowproteolytic activity, so that metabolism of the protein during transitthrough the skin is minimized thereby improving bioavailability.

[0005] One problem with transdermal administration of proteins andpeptides is that they may exhibit very low permeability through the skindue to their hydrophilicity and high molecular weight. One approach toovercoming the low skin permeability is directed to temporarilycompromising the integrity or physicochemical characteristics of theskin to enhance skin penetration, e.g., using a skin penetrationenhancer, employing ultrasonic vibration, removing the epithelial layerby suction or employing an electric current (iontophoresis). Theseapproaches have demonstrated the feasibility of transdermaladministration of proteins and peptides, however are associated withskin irritation and/or other disadvantages.

SUMMARY OF THE INVENTION

[0006] Accordingly, it is an object of the invention to provide acomposition for administration of a protein or peptide transdermally.More specifically, it is an object of the invention to provide acomposition for transdermal administration of a cytokine.

[0007] In one aspect, the invention includes a pharmaceuticalcomposition for dermal or transdermal administration of a cytokine. Thecomposition includes a conjugate composed of a cytokine and at least onefatty acid moiety having between 12-24 carbon atoms covalently attachedto the cytokine. The conjugate has a substantially higher rate of skinpenetration than the cytokine alone.

[0008] In one embodiment, the cytokine is an interferon or aninterleukin, and in a preferred embodiment, the cytokine is interferonα, interferon β, interferon γ, interleukin 1, interleukin 2 orinterleukin 13.

[0009] The fatty acid to which the cytokine is attached is a saturatedfatty acid having between 12-24 carbon atoms or an unsaturated fattyacid having between 12-20 carbon atoms. In preferred embodiments of theinvention, the fatty acid is palmitic acid, behenic acid or lignocericacid.

[0010] One preferred conjugate includes interferon α as the cytokine andpalmitic acid as the fatty acid.

[0011] In another aspect, the invention includes a method for dermal ortransdermal administration of a cytokine. The method includes preparinga conjugate, as described above, and applying the conjugate to the skinof a subject in a pharmaceutically acceptable preparation.

[0012] In another aspect, the invention includes a method of treating aninfection caused by human papilloma virus in a subject by administeringtopically at the site of infection, a conjugate as described above. Inone embodiment of the method, the infection to be treated is genitalwarts and the cytokine in the conjugate is interferon α.

[0013] In another aspect, the invention includes a method of enhancingan immune response to a vaccine, by administrating topically to apatient receiving a vaccine, a conjugate composed of a cytokine and,covalently attached to the cytokine, at least one fatty acid moietyhaving between 12-24 carbon atoms.

[0014] These and other objects and features of the invention will bemore fully appreciated when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows a synthetic reaction scheme for acylation of acytokine;

[0016]FIG. 2 shows a synthetic reaction scheme for acylation ofinterferon with palmitic acid;

[0017]FIG. 3 shows the nucleotide sequence of interferon α2b (SEQ ID No.1)

[0018] FIGS. 4A-4B are capillary electrophoresis electropherogramsshowing the time dependence of derivatization of interferon γ withpalmitic acid (FIG. 4A) and the effect of protein:reagent ratio on thederivatization (FIG. 4B);

[0019] FIGS. 5A-5B are plots of mobility, determined by capillaryelectrophoresis, as a function of cytokine:fatty acid ester ratio (FIG.5A) and time (FIG. 5B) for interferon α2b derivatized with palmitic acid(FIG. 5A) and oleic acid (FIG. 5B, closed triangles);

[0020]FIG. 6A is a chromatographic profile of palmitoylated interferonα2b on Sephadex G-25:

[0021]FIG. 6B is a SDS-PAGE pattern of the corresponding chromatographedfractions of FIG. 6A after silver staining;

[0022]FIG. 6C is a SDS-page profile of palmitoylated interferon α2bsynthesized under various conditions;

[0023] FIGS. 7A-7B are plots showing binding to human keratinocytes ofinterferon α2a as a function of concentration of interferon α2a (FIG.7A) and of interferon α2a derivatized with behenic acid (closed circles)and lauric acid (closed diamonds) and interferon α2a treated with DMSO(closed squares) (FIG. 7B);

[0024]FIG. 8A is a plot showing in vitro percutaneous absorption throughhuman skin as a function of time of conjugates of interferon α2b andpalmitic acid (closed diamonds), oleic acid (open triangles), myristicacid (open diamonds), stearic acid (open circles) and lauric acid (opensquares) and of liposomally-entrapped interferon α2b (closed circles)and interferon α2b alone (closed squares); and

[0025]FIG. 8B is a bar graph showing in vitro cutaneous absorption intohuman skin after 24 hours of the formulations shown in FIG. 8A, whereabsorption into whole skin and into skin after removal of the stratumcorneum is reported for each formulation.

DETAILED DESCRIPTION OF THE INVENTION

[0026] I. Preparation of the Conjugate

[0027] As discussed above, the conjugate of the invention is composed ofa cytokine and a fatty acid moiety covalently attached to the cytokine.As used herein, a cytokine includes any immune system protein that is abiological response modifier. Generally, cytokines coordinate antibodyand T cell immune system interactions and amplify immune reactivity andinclude monokines synthesized by macrophages and lymphokines produced byactivated T lymphocytes and natural killer cells. Monokines includeinterleukin 1, tumor necrosis factor, α and β interferons andcolony-stimulating factors. Lymphokines include interleukins, interferonγ, granulocyte macrophage colony-stimulating factor and lymphotoxin.Cytokines are also synthesized by endothelial cells and fibroblasts.

[0028]FIG. 1 shows a synthetic reaction scheme for derivatizing aprotein, in particular a cytokine, having amino positions available forcovalent attachment, with a fatty acid. In the first step of theprocess, the N-hydroxysuccinimide ester of the fatty acid is prepared bymixing the fatty acid with N-hydroxysuccinimide in a suitable solvent inthe presence of dicyclohexylcarbodiimide. The fatty acid ester is thenisolated by recrystallization or other technique. In the second step,the fatty acid ester is mixed with the protein to react with availableamino groups to yield the fatty acid linked to the protein through anamide bond.

[0029] It will be appreciated that other reaction schemes are suitableto derivatize a protein with a fatty acid. For example, the amide bondformation can be done more selectively by blocking and de-blockingcertain groups on the protein. The protein can also be derivatized withthe fatty acid through formation of an ester bond.

[0030] In studies performed in support of the invention, interferon α,more specifically, interferon α2b, interferon α2a and interferon γ, werederivatized with various fatty acids according to the scheme set forthin FIG. 1. The procedure is suitable for derivatization of otherproteins, such as IL-4, IL-12 and GM-CSF.

[0031] A reaction scheme for fatty acylation of interferon with palmiticacid is illustrated in FIG. 2. Fatty acylation of interferon α by thisreaction forms an amide bond which is stable for dosage form developmentand in biological environments. As described in Example, 1, the firststep in the synthesis is to prepare N-hydroxysuccinimide-palmitate,which, in the second step of the process, is reacted with interferon ina suitable solvent, such as dimethylsulfoxide or dimethylformamide.

[0032] Interferon α2b is a hydrophilic protein with nine lysine aminoacids, which, with reference to FIG. 3, are at positions 31, 49, 70, 83,112, 121, 131, 134 and 164. These lysine amino acids, in addition to theamino terminal, are available for potential covalent attachment of fattyacids. Interferon α2b has disulfide bonds between residues 1 and 19 andbetween residues 29 and 138 (Wetzel, Nature, 289:606, 1981), and onlythe latter disulfide bond is critical for maximal antiviral activity(Morehead, et al., Biochemistry, 23:2500, 1984). Three structurallydistinct domains are important for activity: 10-35, 78-107 and 123-166(Fish, et al., J. Interferon Res., 9:97, 1989).

[0033] As noted above, interferon α has nine lysine residues, as well asthe terminal cysteine, for potential acylation. Depending on theavailability of these positions for acylation and on the reactionconditions, one or more positions can be derivatized with a fatty acid.The three dimensional structure of interferon α has been constructed bycomputer modeling for the primary amino acid sequence of consensusinterferon α (Korn, et al., J. Interferon Res., 14:1, 1994). The modelindicates that the conformationally accessible regions forderivatization within interferon α are domains 29-35, 79-95 and 123-140.Thus, at least the four lysine residues within these regions (positions31, 83, 131 and 134), plus the terminal amino acid, are conformationallyavailable to bind with a fatty acid.

[0034] Because the reaction shown in FIG. 2 is a non-specific acylationsynthesis, it is expected that some of the lysine ε-amino groups and theterminal amino group on the protein will be acylated. The actual fattyacid-derivatized interferon is likely a mixture containing interferon αacylated to various degrees, i.e., mono-palmitate, di-palmitate, etc.For the purpose of the studies reported herein, the different fractionswere not separated or purified. However, it will be appreciated that thefractions can be separated if desired in order to optimize activity andrate of transdermal penetration of the conjugate.

[0035] The degree of derivatization appears to be time dependent, asevidenced by the electropherogram in FIG. 4A. The trace in FIG. 4A wasobtained by capillary electrophoresis and the methodology is set forthin the methods section below. The trace shows that after 2 and 18minutes of reaction time with palmitic acid, the migration time of thepalmitoylated interferon changed from 7 minutes to 7.8 minutes,respectively. Smaller changes in migration time up to 1 hour ofincubation was observed. After 1 hour of reaction time, no furtherchange in migration was observed.

[0036] The effect of protein:N-hydroxysuccinimide ester of palmitic acidratio on palmitoylation was evaluated using capillary electrophoresis.As seen in FIG. 4B, at low ratios of protein:palmitic acid a moreheterogeneous population of derivatized protein was formed, as evidencedby the broader peaks with lower mobility. At a ratio of 1:10 or higher areproducible population of palmitoylated interferon α2b with anelectrophoretic mobility of 9.5 minutes was obtained.

[0037]FIG. 5A is a plot which corresponds to the trace of FIG. 4B andshows the mobility of the interferon (α2b-palmitic acid conjugate as afunction of protein:fatty acid ester (palmitic acid esterified withN-hydroxysuccinimide) ratio. The fatty acid ester has a mobility ofabout 23 and conjugation with interferon α2b at a 1:1 ratio decreasingthe mobility to about 17. The mobility decreases slowly thereafter withincreasing protein:fatty acid ester ratio.

[0038]FIG. 5B shows mobility as a function of time for theN-hydroxysuccinimide ester of oleic-acid (closed triangles) and for theoleic acid-interferon α2b conjugate prepared in a 50/50 v/v mixture ofdistilled water/DMSO and a protein:fatty acid ester ratio of 1:25(closed diamonds). After about 30 minutes of incubation time, themobility of the conjugate is about 17, with a slow continuous decreasein mobility with longer reaction time.

[0039] Further in support of the invention, interferon α2b andinterferon α2a were derivatized as described above with fatty acidshaving between 12 and 24 carbon atoms. The conjugates prepared and themolar ratio of interferon α to the N-hydroxysuccinimide fatty acid esterare shown in Table 1. The mobility values shown in Table 1 weredetermined by capillary electrophoresis, as set forth in the methodssection below. TABLE 1 Fatty Acid Cytokine-Fatty Mobility in Cytokine(No. Carbons) Acid¹ Ratio SDS Gel² Interferon α2b Lauric Acid 1:20  nd³(C12) Interferon α2b Myristic Acid 1:20 nd (C14) Interferon α2b PalmiticAcid 1:20 nd (C16) Interferon α2b Stearic Acid 1:20 nd (C18) Interferonα2b Oleic Acid (C18, 1:20 nd unsaturated) Interferon α2a Lauric Acid1:25 12.532 (C12) Interferon α2a Myristic Acid 1:25 12.533 (C14)Interferon α2a Palmitic Acid 1:25 12.608 (C16) Interferon α2a StearicAcid 1:25 12.636 (C18) Interferon α2a Oleic Acid (C18, 1:25 12.627unsaturated) Interferon α2a Arachidic Acid 1:25 nd (C20) Interferon α2aBehenic Acid 1:25 nd (C22) Interferon α2a Lignoceric Acid 1:25 nd (C24)Interferon α2a none — 13.085 (control) Interferon α2a in none — 13.213DMSO (control)

[0040] II. Characterization of the Conjugates

[0041] The conjugates composed of interferon α and various fatty acids,prepared as described above, were characterized by electrophoresis(polyacrylamide gel electrophoresis (PAGE)) and were characterized forantiviral activity and receptor binding activity.

[0042] 1. Gel Electrophoresis

[0043] A chromatographic profile of interferon α2b acylated withpalmitic acid on Sephadex G-25 column is shown in FIG. 6A. Theintactness of the interferon α2b after lipid modification is evident andthe individual column (Sephadex G25) fractions are shown in the SDS-PAGEpattern of FIG. 6B. Lane 1 in the profile is for a Bio-Rad molecularweight standard; lane 2 is for an interferon α2b standard and lanes 3-9correspond to fractions taken at 1.5-5.5 ml from the Sephadex column(FIG. 6A).

[0044]FIG. 6C is a SDS-PAGE profile comparing interferon α2b-palmitateconjugates prepared under various conditions. Lane 1 in the profile is amolecular weight standard; lane 2 is interferon α2b incubated in DMF;lane 3 corresponds to a conjugate of interferon α2b and palmitic acidprepared in DMF; lane 4 corresponds to interferon α2b incubated in DMSO;lane 5 corresponds to a conjugate of interferon α2b and palmitic acidprepared in DMSO; lane 6 is an interferon α2b standard, 100 ng; and lane7 is an interferon α2b standard, 50 ng.

[0045] A comparison of the bands in lanes 3 and 5 shows that the yieldof palmitoyl-interferon α2b prepared in DMSO was 15-20% higher than whenthe conjugate was prepared in DMF. Lanes 2 and 4 in FIG. 6C compare theeffect of the two solvents, DMSO and DMF, respectively, on the proteinalone. No differences in the bands are apparent, indicating that theneither solvent has a negative effect on the protein. The PAGE bands forthe conjugate indicate a 6-10% increase in molecular weight ofinterferon α after acylation.

[0046] 2. Antiviral Activity

[0047] The palmitate-interferon α2b conjugate prepared as describedabove was evaluated for antiviral activity to determine whether acylatedcytokines in general retain biological activity. Antiviral activity wasevaluated according the procedure described in Example 2, where thecytopathic effect inhibition assay using Georgia Bovine Kidney (GBK)cells and vesicular stomatitis virus (VSV) as the challenge virus. Theresults are shown in Table 2. TABLE 2 Antiviral Activity (% ofinterferon-α2b) conjugate prepared conjugate prepared in DMSO¹ in DMF¹Interferon α2b¹ 100% 100% palmitoyl-interferon α2b  50%  0%

[0048] The antiviral activity of interferon α2b was unaffected when theprotein was treated to the conditions of the acylation reaction, exceptfor addition of palmitic acid, in both dimethylformamide (DMF) anddimethylsulfoxide (DMSO). That is, 100% of the antiviral activity ofinterferon α was preserved. Acylation of the cytokine with palmitic acidin the solvent DMF resulted in a complete loss of activity. When thereaction was carried out in DMSO a 50% preservation of antiviralactivity was achieved.

[0049] The loss in activity may be in part attributed to experimentalconditions, and the assay was modified for greater control and accuracy.The GBK cells in 96-well microtiter plates were dosed with 50 μlinterferon α2b reference solution of a conjugate sample. Afterincubation overnight the cells were infected with VSV virus. Afterincubation, washing, fixing and staining, the plates were read by aspectrophotometer to determine the antiviral activity of the compounds.The results, shown in Table 3, indicate enhanced activity of the novelderivatives compared to the parent protein. TABLE 3 Sample AntiviralActivity Interferon α2b 100% (INF α2b) Lauroyl-INF α2b 210% Myristol-INFα2b 175% stearoyl-INF α2b 190% oleyl-INF α2b 200%

[0050] In another experiment using the revised method, antiviralactivity of interferon α2a derivatized with behenic and lignoceric acidwas measured. The conjugate including behenic acid retained nearly 100%of the interferon α2a activity and the conjugate with lignoceric acidretained about 30% of interferon α2a antiviral activity.

[0051] Table 4 shows the antiviral activity of conjugates prepared withinterferon γ. TABLE 4 Antiviral Activity Fatty Acid (% of interferon γ)Lauric Acid 25% Myristic Acid 20% Palmitic Acid 22% Stearic Acid 40%Oleic Acid 10% Arachidic Acid  2% Behenic Acid  8% Lignoceric Acid  9%

[0052] As noted above, the conjugates used in the studies reportedherein were not separated or purified into single acyl-proteinfractions. There may be an optimum degree of fatty acylation for maximumretention of biological activity of the cytokine—for example, adi-palmitoyl interferon α may have a higher, or lower, biologicalactivity than tri-palmitoyl interferon α. Separation of the fractionsfor analysis can be readily performed by those of skill in the art todetermine such an optimum, as evidenced by the work of Hashimoto, et al(Pharm. Res., 6:171, 1989). Nonetheless, partial loss of antiviralactivity does not exclude the possibility that other functions ofinterferon α are unchanged or perhaps increased. In fact, some cytokinefunctions do not involve receptor binding and can act directly onintercellular signaling pathways (Baron et al., JAMA, 266:1375, 1991).Also, partial loss of antiviral activity may be inconsequential or atleast offset in view of the enhanced skin penetration, discussed below.

[0053] 3. Receptor Binding

[0054] Binding of the conjugates composed of interferon α2a and behenicacid or lauric acid was determined in an assay using humankeratinocytes, as described in Example 3. The results are shown in FIGS.7A-7B, where FIG. 7A shows binding of iodinated interferon α2a to humankeratinocytes as a function of concentration of interferon α2a. Thebinding of interferon α2a is concentration dependent and saturation ofbinding was not evident at 40 ng interferon α2a. Scatchard analysisindicated the dissociation constant was 5.1×10⁻¹⁰M, with 1579 receptorsper human keratinocyte cell (see insert in FIG. 7A).

[0055]FIG. 7B shows binding of conjugates of interferon α2a derivatizedwith behenic acid (closed circles) and lignoceric acid (closed circles)and, as a control, of interferon α2a treated with DMSO (closed squares)as a function of amount of interferon α2a. The behenic acid-interferonα2a conjugate had a binding comparable to that of the protein alonetreated with DMSO.

[0056] 4. Solubility

[0057] The relative hydrophobicity of the conjugates described abovewere determined by measuring the partition coefficient of each conjugateinto stratum corneum. Powdered stratum corneum, prepared as described inExample 4, was incubated with radiolabeled interferon α2a and the lipidderivatized conjugates, prepared as described above, and the ratio ofuptake (Kp) into the powdered stratum corneum to that remaining in thesaline preparation was determined (Example 4). The results are shown inTable 5. TABLE 5 Conjugate Kp interferon α2a 3.360 lauricacid-interferon α2a 4.404 myristic acid-interferon α2a 4.541 palmiticacid-interferon α2a 5.071 stearic acid-interferon α2 4.508 oleicacid-interferon α2a 5.044 arachidic acid-interferon α2a 5.079 behenicacid-interferon α2A 3.555 lignoceric acid-interferon α2a 3.730 DMSOtreated interferon α2a 3.906

[0058] The results show that the fatty acid derivatization of interferonincreases the uptake relative to the parent protein, indicating anincrease in hydrophobicity and greater affinity for the skin.

[0059] A similar study was conducted for interferon α2b and conjugatesof interferon α2b, where the partition coefficient was determined in theconventional octanol/water system, at octanol/phosphate buffered salineratios of 1:7 and 1:25. The results are shown in Table 6. TABLE 6 pvalue (paired Test System Conjugate Kp t-test) octanol/saline (1:7)interferon α2b 0.0348 lauric acid-interferon α2b 0.0737 0.103 myristicacid-interferon α2b 0.0691 0.001 palmitic acid-interferon α2b 0.03640.800 stearic acid-interferon α2b 0.0531 0.024 oleic acid-interferon α2b0.0329 0.540 octanol/saline (1:25) interferon α2b 0.0373 lauricacid-interferon α2b 0.0434 0.423 myristic acid-interferon α2b 0.04870.201 palmitic acid-interferon α2b 0.0337 0.634 stearic acid-interferonα2b 0.0263 0.142 oleic acid-interferon α2b 0.0475 0.265

[0060] 5. Cutaneous Absorption

[0061] The rate and extent of skin penetration of the conjugates wasdetermined in vitro according to the procedure described in Example 5.In these studies, interferon α2b and the palmitoyl derivative ofinterferon α2b were iodinated by the lactoperoxidase method set forth inExample 3. A preparation of each test compound was placed on fullthickness human skin mounted in a diffusion cell and the downstreamreservoir of the cell was monitored for 24 hours for amount ofinterferon α2b.

[0062] After 24 hours, the skin was removed from the test cells and theradioactivity associated with the skin was determined by gamma counting.These results are shown in Table 7 under the column headed “whole skincounts”. The skin was then stripped ten times with Scotch™ tape and theradioactivity associated with each strip was determined separately.These values are reported in Table 7 in the column headed “stratumcorneum”. The skin after stripping was counted again to obtain thecounts associated with the viable skin layers (epidermis, dermis andsubcutaneous tissues), and this data is in the third column of Table 7.The skin stripping technique was validated by sectioning paraffinembedded stripped skin and viewing under a light microscope for completeremoval of the stratum corneum. TABLE 7 In vitro cutaneous absorption ofinterferon α2b and palmitoyl-interferon α2b into human breast skinStratum Whole Skin Corneum Viable Layers Preparation μg/cm², n = 6μg/cm², n = 6 μg/cm², n = 6 interferon α2b 0.41 ± 0.11 0.20 ± 0.08 0.23± 0.09 (1.8% ± 0.5%) (0.98% ± 0.39%) palmitoyl- 2.11 ± 1.22 0.23 ± 0.141.88 ± 1.16 interferon α2b (11.5% ± 6.7%) (10.3% ± 6.4%)

[0063] The results in Table 7 show that both the cutaneous andpercutaneous absorption of the acylated cytokine was 5-6 fold greaterthan that of the cytokine alone. The amount of acylated interferon α2band of interferon α2b in whole skin after 24 hours of treatment was2.11±1.22 μg/cm² and 0.41±0.11 μg/cm², respectively. This represents11.5±6.7% and 1.8%±0.5% of total drug applied, respectively. In theviable skin layers the difference in absorption between the derivatizedprotein and the parent protein was 8-10 fold, 1.88±1.16 μg/cm²(10.3%±6.4%) and 0.228±0.91 μg/cm² (0.98%±0.39%).

[0064] The calculated percutaneous absorption parameters for thepreparations reported in Table 7 are shown in Table 8. Approximately twotimes higher flux was detected for the conjugate compared to thenon-fatty acylated protein. The total amount of drug diffused in 24hours was also about two times higher for the conjugate. TABLE 8 Invitro percutaneous Absorption of interferon α2b and palmitoyl-interferonα2b through human breast skin ¹²⁵I-interferon ¹²⁵I-palmitoyl- Parametersα2b interferon α2b Steady state flux (ng/cm²/h)¹ 1.47 2.71 Permeabilitycoefficient (cm/h)² 1.65 × 10⁻⁵ 3.03 × 10⁻⁵ Diffusion coefficient(cm²/sec)³ 6.85 × 10⁻¹² 5.45 × 10⁻¹² Total amount diffused in 24 h: 23.8± 17.4 42.7 ± 25.70 (ng/cm²)

[0065] The cutaneous and percutaneous absorption into and through skinwas also measured in vitro for conjugates of interferon α2b and lauricacid, myristic acid, palmitic acid, stearic acid and oleic acid,prepared as described above. A preparation of liposomes having entrappedinterferon α2b was also tested. The results are shown in FIGS. 8A-8B,where in FIG. 8A the amount of interferon α2b absorbed percutaneously isreported, e.g., the quantity of interferon α2b in the downstreamreceiving volume after 24 hours. FIG. 8B shows the amount of interferonα2b in the skin after 24 hours.

[0066] The figures show that fatty acylation of the cytokine enhancedpercutaneous absorption significantly when compared toliposomally-entrapped interferon α2b (closed circles) and interferon α2balone (closed squares). As seen in FIG. 8A, the conjugate with palmiticacid (closed triangles) had the highest percutaneous absorption,followed by oleic acid (open triangles), myristic acid (open diamonds),stearic acid (open circles) and lauric acid (open squares). Interferonα2b entrapped in liposomes (closed squares) and the control formulationof interferon α2b alone (closed squares) had the lowest cutaneouspenetration rates.

[0067]FIG. 8B is a bar graph showing the amount of interferon α in wholeskin and in the viable skin, that is, skin after removal by tapestripping of the stratum corneum for the formulations with interferonα2b shown in FIG. 8A and for two formulations with interferon α2a;behenic acid-interferon α2a and lignoceric acid-interferon α2a.

[0068] The in vitro skin penetration results show that fatty acylationof a cytokine is effective to significantly increase the skinpenetration of the cytokine. “Significantly is increase” or“substantially higher rate of skin penetration” as used herein meansthat the skin penetration, that is cutaneous or transcutaneouspenetration, is increased by at least two-fold, more preferablythree-fold, over the skin penetration of the cytokine alone.

[0069] III. Method of Use

[0070] In another aspect, the invention includes a method oftransdermally delivering a cytokine by preparing a conjugate of thecytokine as described above and applying the conjugate to the skin. In apreferred embodiment of this aspect, the conjugate is composed ofinterferon α and a fatty acid having between 12-24 carbon atoms and isadministered topically for treatment of genital warts caused by humanpapilloma virus.

[0071] The conjugate is typically applied to the skin in apharmaceutically acceptable preparation, by which is meant anypreparation or device suitable for maintaining the conjugate in contactwith the skin. For example, such a preparation can be a simple salinesolution containing the conjugate that is gelled with a suitable gellingagent, such as a cellulose derivative, to a viscosity suitable forapplication. In general, topical gels, creams or ointments arepreferred, however non-rate limiting transdermal devices that canincorporate the conjugate are also suitable.

[0072] Preferred cytokines for use in the invention are interferons andinterleukins. The interferons are a group of immunoregulatory proteinssynthesized by T lymphocytes, fibroblasts and other types of cellsfollowing stimulation with viruses, antigens, mitogens, double-strandedDNA or lectins. Interferons have immunomodulatory functions and enhancethe ability of macrophages to destroy tumor cells, viruses and bacteria.Interferons are classified as α and β, which have antiviral properties,and as γ which is known as immune interferon. The α and β interferonsshare a common receptor, and γ interferon has its own receptor.Interferons α and β are synthesized mainly by leukocytes and fibroblastsand are acid stable. Interferon γ is acid labile and is formed mainly byT lymphocytes stimulated by antigen or mitogen, but is also secreted bynatural killer cells.

[0073] The ability of interferons to prevent infection of noninfectedcells is species specific, it is not virus specific. Essentially allviruses are subject to the inhibitory action of interferons. Interferonsinduce formation of a second inhibitory protein that prevents viralmessenger RNA translations.

[0074] Interferon α2b (recombinant) is a 18.4 kDa molecular weightpolypeptide consisting of 165 amino acids. Interferon α shows multiplebiological effects including antiviral, antiproliferative andimmunomodulatory. The mechanism of action is through binding to specificcell surface receptors. The binding induces protein kinase and2′5′-oligoadenylate synthetase (Clemens, Br. J. Clin. Pract., 42:5,1988). These enzymes can inhibit protein synthesis in the cell andtherefore can prevent a virus from replicating (Pestka, et al., Ann.Rev. Biochem., 56:727, 1987).

IV. EXAMPLES

[0075] The following examples illustrate methods of preparing,characterizing, and using the acylated cytokine conjugate of the presentinvention. The examples are in no way intended to limit the scope of theinvention.

[0076] A. Materials

[0077] Interferon α2b was provided by Schering-Plough ResearchCorporation, Kenilworth, N.J. Interferon α2a and interferon γ wereobtained from (Roche Biosciences). The fatty acids lauric acid, myristicacid, palmitic acid, stearic acid, arachidic acid, lignoceric acid,oleic acid and behenic acid were obtained from Sigma Chemical Co. (St.Louis, Mo.). N-hydroxysuccinimide was obtained from Sigma Chemical Co.

[0078] B. Methods

[0079] 1. Page

[0080] Polyacrylamide gel electrophoresis (PAGE) in the presence ofsodium dodecyl sulfate (SDS) was carried out in a Mini-Protean II(BioRad, Missisauga, Ontario, Canada) apparatus according to Laemmli(Nature, 227:680, 1970). The gel consisted of a running gel containing14% (w/v) acrylamide and a stacking gel containing 5-acrylamide. The gelthickness was 1.0 mm. The electrophoresis buffer was 25 mM Tris, 192 mMglycine, 0.01% (w/v) SDS, pH 8.6. Electrophoresis was carried out at 200V constant voltage. The electrophoresis was conducted for 45 minutes.After electrophoresis, the gels were silver stained to detect theprotein (Foldvari, et al., Biochem Cell Biol., 68:499, 1990).

[0081] 2. Capillary Electrophoresis

[0082] Capillary electrophoresis studies were performed using a P/ACESystem 5500 (Beckman Instruments, Fullerton, Calif.) with diode arraydetector and System Gold Software. Free-zone electrophoresis was carriedout using an uncoated capillary (57 cm×75 μm) at 23° C. and 20 KV with a5 second pressure injection. The running buffer was 0.6% w/v sodiumborate (Na₂B₄O₇.10H₂O) and 0.5% boric acid, pH 8.75. The detector wasused at 200-300 nm. Prior to use, the capillary was washed with NaOH(0.1M) for 10 minutes and for 1 minute between each run.

Example 1 Preparation of Palmitoyl Derivative of Interferon α2b

[0083] Palmitoyl derivatives of interferon α2b were synthesizedaccording to the scheme shown in FIG. 2, where the N-hydroxysuccinimideester of palmitic acid (NHS-P) was synthesized as follows. Equal molaramounts of palmitic acid and N-hydroxysuccinimide were mixed together inethyl acetate followed by addition of dicyclohexylcarbodimide (DCI). Themixture was stirred overnight at 4° C. Dicyclohexylurea was filtered outand NHS-P was recrystallized from the filtrate by the addition ofethanol at 4° C. ¹H-NMR studies on NHS-P confirmed the expectedstructure (results not shown).

[0084] The palmitoyl derivative of interferon α2b was prepared follows.NHS-P was dissolved in DMF or DMSO and added at 25:1 molar ratio to thePBS buffer (7.5 mM Na₂HPO₄, 2.5 mM NaH₂PO₄, 151.2 mM NaCl) containinginterferon α2β at pH 7.2. The mixture was kept at room temperature for 3hours with occasional gentle agitation. After the reaction, DMF or DMSOwas removed under vacuum and the residue was redissolved in steriledistilled water.

[0085] The palmitoyl-interferon α2b derivative was separated from freefatty acid by chromatography on Sephadex G-25 column (Pharmacia,Uppsala, Sweden). The yield of palmitoyl-interferon α2b was dependent onthe starting concentration, where a 25 μg batch and a 100 μg batchyielded 50.2% and 84.0%, respectively, as determined by the densitometryof the palmitoyl-interferon bands of the column fractions. Fractionscontaining protein were pooled, freeze-dried and reconstituted withsterile distilled water before use.

[0086] A portion of each fraction was used for polyacrylamide gelelectrophoresis (PAGE) and silver staining according to the proceduredescribed above in the Methods section, and SDS-PAGE profiles ofpalmitoyl-interferon α2b are shown in FIGS. 6A-6B.

Example 2 Antiviral Activity of the Conjugate

[0087] Antiviral activity of palmitoyl derivatives of interferon -a wasdetermined by the cytopathic effect inhibition assay using GeorgiaBovine Kidney (GBK) cells, which are sensitive to human interferon α,and vesicular stomatitis virus (VSV) as the challenge virus (Ohmann, etal., J. Gen. Virol., 65:1485, 1984). The reference standard wasinterferon α2b, specific activity 2.24×10⁸ IU/mg. The results are shownin Table 2.

Example 3 Conjugate Receptor Binding

[0088] A. Iodination of Interferon

[0089] Iodination of interferon α and conjugates of interferon wascarried out using the lactoperoxidase method (Sarkar, et al., MethodsEnzymol., 119:263, 1986). Briefly, 2 mCi ¹²⁵I, obtained from AmershamCorporation (Oakville, Ontario, Canada), was neutralized by adding 3volumes of 0.03 N HCl and the total was made up to 25 μl with 0.2 Msodium phosphate buffer pH 7.2. The following were added to the mixture:50 μl Enzymobeads (Bio-Rad), 15 μl freshly made 2% β-D-glucose in 0.1 Msodium phosphate buffer, pH 7.2, 10 μl interferon (approximately 10 μgprotein) The reaction mixture was incubated for 20 minutes at roomtemperature. The reaction was stopped by adding 25 μl of 1 M sodiumazide and incubating for 15 minutes. Finally, 125 μl of saturatedL-tyrosine in PBS was added and the mixture transferred onto a SephadexG25 column. Fractions containing the protein were pooled.

[0090] In another method, the iodination mixture was transferred ontoBio-Spin columns (exclusion limit 6,000) (Bio-Rad) and iodinated proteinrecovered by a brief low speed centrifugation. To remove any possibleresidue of unbound iodine the protein preparation was dialyzed overnightagainst 1 mM sodium iodide in PBS. This procedure removed practicallyall acid soluble iodine as determined by trichloroacetic acidprecipitation.

[0091] The final preparations of ¹²⁵I-interferon α2A and¹²⁵I-palmitoyl-interferon a had specific activities of 2.05×10⁷ cpm/μgand 1.94×10⁷ cpm/μg protein, respectively. The iodinated interferon αand palmitoyl-interferon α were examined by PAGE for intactness, and theprotein concentration was determined by densitometry.

[0092] B. Receptor Binding

[0093] A single cell suspension of human keratinocyte cells (isolatedfrom patients undergoing mammoplasty within one day of surgery) from aconfluent culture was prepared and resuspended at 2×10⁶ cells/mL inKSF-medium. Two mL of KSF-medium was added to each well of a 6-well flatbottom tissue culture plate and incubated at 37° C. until the cells ineach well reached confluency. ¹²⁶I-interferon α2a conjugates, preparedas described above, at concentrations between 0.5-40 ng and incubated at4° C. for 5 hours on a shaker. The medium was aspirated from each wellto gamma counting tubes and washed three times with 1 mL of cold HBSS.The ¹²⁵I-interferon sample wells were scraped using cell scrapers andexamined using an inverted microscope. The cell suspension wastransferred to the gamma counting tubes and the wells were washed threetime with 0.5 mL of HBSS and transferred to the same tubes. One mL ofHBSS was added to every well to wash the wells and the HBSS wastransferred to another tube. The radiation of each tube was countedusing a gamma counter. The cells in the cell control well were detachedusing 0.25% trypsin. The cells were counted and evaluated to detectviability by trypan blue exclusion. The results are shown in FIGS.7A-7B.

Example 4 Measurement of Partition Coefficients

[0094] Human skin was cut into 1×1 cm squares and placed into 60° C.water for 1 minute. The epidermis was separated with forceps. The peeledskin pieces were placed epidermis side down on filter paper saturatedwith 1% trypsin solution and incubated at room temperature for 1 hour.Then the digested epidermis was washed with water. The stratum corneumpieces were blot dried with tissue and further dried in a freeze dryerovernight. The stratum corneum pieces were ground to form powder usingliquid nitrogen. The portion that can pass through a 60-mesh but not80-mesh sieve was collected for partition coefficient determination.

[0095] Five milligrams of powdered stratum corneum was weighed into eachvial. Fifty μl of fatty acid derivatized ¹²⁵I-interferon α in phosphatebuffered saline was added to cover the stratum corneum. Empty vialswithout powdered stratum corneum were used as controls. The mixture wasincubated for 24 hours at 32° C. with gently shaking followed bycentrifugation at 14,000 rpm for 5 seconds. The supernatant was countedby gamma counting. The powder was washed three times by adding 50 μlphosphate buffered saline. After washing, the stratum corneum powderleft in the vial was counted.

[0096] The partition coefficient (Kp) was calculated as the ratio of(cpm_(psc)/weight of psc)/(cpm_(PBS)/volume of PBS) (psc=powderedstratum corneum; PBS=phosphate buffered saline). The values are shown inTable 5.

Example 5 In Vitro Cutaneous and Percutaneous Absorption

[0097] The rate of diffusion of palmitoyl-interferon α2b across fullthickness human breast skin (freshly obtained from plastic surgery andkept at −20° C. until used within 1 week) was investigated usingTeflon®, Flow-Thru Diffusion Cells (Crown Glass Co. Inc., Somerville,N.J.) (Bronaugh and Stewart, J. Pharm. Sci., 74:64 1985), which have asurface area for diffusion of 0.32 cm². The diffusion cells wereoperated with a continuous perfusion fluid flow of PBS pH of 7.2 on thedownstream side in order to maintain sink conditions. The flow rate ofthe perfusion fluid was 3 mL per hour.

[0098] The diffusion cells were mounted in a diffusion cell heater(Crown Glass Co. Inc.) to maintain the temperature at 37° C. withcirculating water. Each cell was connected to a fraction collector andeach experiment was conducted for a continuous period of 24 hours overwhich time samples were collected at intervals.

[0099] The test preparations consisted of 0.1 mL solution [PBS buffer]or 0.1 g methylcellulose 1500 cP [2.5%] gel hydrated with PBS labeledwith ¹²⁵I-palmitoyl-interferon α2b. The test preparations were instilledinto the cells at concentration of 20×10⁶ IU (89.3 μg) ofpalmitoyl-interferon α2b per g or mL product. The average amount ofinterferon applied was 20.7 μg/cm² skin surface area. The quantity ofpalmitoyl-interferon α2b in the collected fractions was determined bygamma counting and the results are shown in Table 7.

[0100] After 24 hours, the skin was removed from the diffusion cell andrinsed thoroughly with cold (4° C.) PBS (3×15 mL) and the skin wasblotted with tissue paper. The skin surface was swabbed with a cottontip applicator dipped into PBS containing 0.5% Tween 80 two times toremove surface-bound drug. Care was taken not to disturb the stratumcorneum. The skin was carefully folded (epidermal sides together) toavoid contamination of dermal side and placed into glass tubes. Theradioactivity associated with the skin was determined by gamma countingand was considered to be the “whole skin” counts. The skin was thenstripped ten times with a Scotch tape and the radioactivity associatedwith each strip was determined separately. The skin after the strippingwas counted again in a clean tube to obtain the counts associated withthe viable skin (epidermis, dermis and subcutaneous tissue). The skinstripping technique was validated by sectioning the paraffin embeddedstripped skin to observe the complete removal of the stratum corneum inthe light microscope (results not shown).

[0101] Trichloroacetic acid (TCA) precipitation was used to determinefree and bound iodine label in percutaneous fractions and skinhomogenate prepared from treated skin samples. TCA was added to eachsample to 5% w/v concentration and was incubated at 4° C. overnight. Thesupernatants and pellets were analyzed by gamma counting aftercentrifugation in a Beckman Microfuge at 14,000 rpm for 15 minutes. Theexperiments with TCA precipitation from skin homogenates (after tapestripping) and fractions showed that 40-50% of radioactivity wasprecipitated from both interferon α2b and palmitoyl-interferon α2b,indicating that protein, not just the free iodine label, was present.The results are shown in Table 7.

[0102] Although the invention has been described with respect toparticular embodiments, it will be apparent to those skilled in the artthat various changes and modifications can be made without departingfrom the invention.

1 1 1 165 PRT Homo sapiens 1 Cys Asp Leu Pro Gln Thr His Ser Leu Gly SerArg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser LeuPhe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu GluPhe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His GluMet Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser AlaAla Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu TyrGln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly ValThr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val ArgLys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Asp Lys TyrSer Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg SerPhe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser LysGlu 165

It is claimed:
 1. A pharmaceutical composition for dermal or transdermaladministration of a cytokine, comprising a conjugate composed of acytokine and at least one fatty acid moiety having between 12-24 carbonatoms covalently attached to the cytokine, said conjugate having asubstantially higher rate of skin penetration than the cytokine alone.2. The composition of claim 1 , wherein said cytokine is selected fromthe group consisting of interferons and interleukins.
 3. The compositionof claim 1 , wherein said cytokine is selected from the group consistingof interferon α, interferon β, interferon γ, interleukin 1, interleukin2 and interleukin
 13. 4. The composition of claim 1 , wherein said fattyacid is a saturated fatty acid having between 12-24 carbon atoms.
 5. Thecomposition of claim 1 , wherein said fatty acid is an unsaturated fattyacid having between 12-20 carbon atoms.
 6. The composition of claim 1 ,wherein said fatty acid is selected from palmitic acid, behenic acid andlignoceric acid.
 7. The composition of claim 3 , wherein said fatty acidis palmitic acid.
 8. The composition of claim 1 , wherein said cytokineis an interferon α and said fatty acid is palmitic acid.
 9. A method fordermal or transdermal administration of a cytokine, comprising preparinga conjugate composed of said cytokine and, covalently attached to thecytokine, at least one fatty acid moiety having between 12-24 carbonatoms, said conjugate having a substantially higher rate of skinpenetration than the cytokine alone, and applying said conjugate to theskin of a subject in a pharmaceutically acceptable preparation.
 10. Themethod of claim 9 , wherein said cytokine is an interferon or aninterleukin.
 11. The method of claim 10 , wherein said cytokine isselected from the group consisting of interferon α, interferon β,interferon γ, interleukin 1, interleukin 2 and interleukin
 13. 12. Themethod of claim 9 , wherein said fatty acid is a saturated fatty acidhaving between 12-24 carbon atoms.
 13. The method of claim 12 , whereinsaid fatty acid is selected from palmitic acid, behenic acid andlignoceric acid.
 14. The method of claim 9 , wherein said fatty acid isan unsaturated fatty acid having between 12-20 carbon atoms.
 15. Themethod of claim 9 , wherein said cytokine in interferon α and said fattyacid is palmitic acid.
 16. A method of treating an infection caused byhuman papilloma virus in a subject, comprising administering topicallyat the site of infection, a conjugate composed of said cytokine and,covalently attached to the cytokine, at least one fatty acid moietyhaving between 12-24 carbon atoms, said conjugate having a substantiallyhigher rate of skin penetration than the cytokine alone.
 17. The methodof claim 16 , wherein said infection is genital warts and said cytokineis interferon α.
 18. A method of enhancing an immune response to avaccine, comprising administrating topically to a patient receiving avaccine, a conjugate composed of a cytokine and, covalently attached tothe cytokine, at least one fatty acid moiety having between 12-24 carbonatoms, said conjugate having a substantially higher rate of skinpenetration than the cytokine alone.